4.2 Microbiome of Ceratitis capitata
Despite a wide range of host plants and large distances among collection sites, many microbial taxa were identified in both the native and introduced ranges of the medfly. The microbiome composition was highly similar across the biological replicates and the sampling sites, except for the specimens collected in Brazil. The microbiome of all samples was dominated by the phylum Proteobacteria followed by a few other predominant phyla: Firmicutes, Bacteroidetes, and Actinobacteria, which is consistent with the significant phyla previously observed in association with tephritids (Deutscher, Chapman, Shuttleworth, Riegler, & Reynolds, 2019; Morrow, Frommer, Royer, Shearman, & Riegler, 2015; Nikolouli et al., 2020).  It is important to note that in this study, we used individual mature adults collected in wild conditions, while previous studies were performed on pooled dissected guts of larvae (De Cock et al., 2019; De Cock et al., 2020), adult guts (Nikolouli et al., 2020) or adults collected from infected fruits that emerged in laboratory conditions (Malacrino et al., 2018). Despite the technical differences, these studies have defined Proteobacteria  as the predominant phylum, albeit at a higher abundance (≥ 80%) compared to our study (60.4%). Besides this, the abundance of the phylum Firmicutes in those studies was lower (≤ 10%) compared to our results (18.7%). Differences observed in abundances for these phyla might be explained by differences in the sample source and/or life stage of the medflies analysed. At different taxonomic levels, the prevalence of an unclassified Enterobacteriaceae genus  (Gammaproteobacteria) was expected because it is known to dominate the first set of maternally inherited microbiota, which is vertically transmitted during oviposition in tephritids (Aharon et al., 2013; Behar, Yuval, & Jurkevitch, 2005; Deutscher et al., 2019).
The microbiome abundance and composition remained similar to the native range across different host plants and some distant localities despite the low number of ASVs detected in South Africa. Furthermore, the microbiome structure in the introduced range presented similar numbers of ASVs among Spain, Israel, Australia and Colombia, with significant differences in their microbiome composition only found for Spain and Israel. These results of the microbiome structure mirror our population genetic analysis, where we found genetic similarities among all the populations in the introduced range, except Brazil. Therefore, we suggest that these microbial communities are stable regardless of the distances, host plants and differences in environmental conditions, also reflecting the interconnectivity between these localities. In this context, similarities in microbiome composition were found between Spain and Australia, which were also highly connected populations in our DAPC (both populations appeared in the same genetic cluster) and population structure analysis. On this basis, the connectivity of the medfly populations might facilitate a bacterial exchange/transmission across them, suggesting the attainment of an essential microbiome set over time that successfully serves the medfly’s polyphagous nature (Gruber et al., 2019). This could represent a solid contributing factor to the species’ invasive success by ensuring a universal way of feeding on various plants in introduced ranges.
A particular case is the unique microbiome composition in the Brazilian population. A recognised factor that influences microbiome composition is feeding on different plant species (Malacrino et al., 2018). However, in this study, Brazil and the native South Africa were collected from the same host plant, Guava. Consequently, we suggest that the somewhat isolated flies of Brazilian populations acquired new bacteria from the new environment. Also, exclusively in Brazil, we found in high relative abundance Acinetobacter  (phylum Proteobacteria) that was previously described in larvae of medfly (De Cock et al., 2019; Malacrino et al., 2018). The genus Acinetobacter  is associated with plant defence suppression mechanisms in polyphagous insects, which is known to help insects to detoxify phenolic glycosides in vitro(Mason, Couture, & Raffa, 2014), although some other bacteria, which are difficult to isolate in culture, may contribute to the metabolism of this metabolite.
Unclassified Burkholderiaceae in Brazil were previously described only in medfly adults collected in Italy (Malacrino et al., 2018). The presence of these bacteria has been associated with nitrogen fixation (i.e. diazotroph microbes), which is an essential mechanism for the fly’s nutrition, development, and reproduction (Behar et al., 2005; Raza, Yao, Bai, Cai, & Zhang, 2020). Most insect microbiomes are maintained by strict vertical transmission, however, noteworthy is the case of the bean bug Riptortus pedestris (Heteroptera: Alydidae), where it is known that some strains of Burkholderia are taken at early stages every generation from the environment (Kikuchi, Hosokawa, & Fukatsu, 2007). Members of the genus Burkholderia  are known as significant soil bacteria, although the details of the acquisition mechanisms of the bacteria in R. pedestris  remain unknown (Kikuchi et al., 2007; Kikuchi, Meng, & Fukatsu, 2005). However, in agricultural lands with intensive insecticide applications, an acceleration in the microbial degradation of the insecticide has been observed (Arbeli & Fuentes, 2007; Singh, Walker, & Wright, 2005). Subsequently, when R. pedestris  occurs in fields heavily treated with the insecticide fenitrothion (one of the most popular organophosphates), some Burkholderia  strains show the ability to degrade the insecticide and demonstrate that Burkholderia  confers resistance to the insect against the organophosphate, establishing a beneficial symbiont relationship (Kikuchi et al., 2012; Kikuchi & Yumoto, 2013). This finding raises the possibility that the Burkholderia  observed only in Brazilian medflies may be associated with the novel acquisition of capabilities to hydrolyse and metabolise insecticides, thus enhancing the fitness of the host insect. Although this hypothesis should be further tested, it might be an example of horizontal transmission of microbiomes from the soil associated with a demographic response to insecticides and calls for specific experiments to understand the emergence of resistance and the bacterial strains involved. Another example to consider as horizontal transmission observed exclusively in Brazil  is Dysgonomonas.  This member of phylum Bacteroidetes is found on the surface of plant roots in the soil (Liu et al., 2018) and has been described in the guts of wild specimens ofBactrocera dorsalis,  a species closely related to the medfly, suggesting that they might have been recruited from the surrounding environment (Wang, Jin, & Zhang, 2011).
Overall, most of the prevalent genera identified exclusively in Brazil are associated with the soil microbiome, which raises questions about the possible functions of the microbiome in this specific locality. A recent publication found differences in chromosomes and changes in the allele frequency between experimental and natural populations of D. melanogaster  that were exposed to different microbiome treatments, suggesting that a shift in microbiome composition may be an agent of selection that drives adaptation at population levels (Rudman et al., 2019). Here, we reveal the correlation between the microbiome composition and the genomic structure of the populations and highlight the importance of the host-microbiome interaction for the adaptation of the medfly to different environments. Therefore, further research at a population level will be required to unveil the role of the microbial communities in the medfly.